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BACKGROUND This study developed a feasible catalytic method for d-allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers. RESULTS d-Allulose 3-epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo-inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing twelve residues (GSAGSAAGSGEF). Under the conditions of 55 °C and pH 8.0 as well as with the addition of 1 mM Mn2+ , the CSCA-linkerE-DAE fusion enzyme obtained through high cell density cultivation displayed a maximal exo-inulinase activity of 21.8 U mg-1 and resulted in a yield of 6.3 g L-1 d-allulose and 39.2 g L-1 d-fructose using 60 g L-1 inulin as the raw material. Catechol-modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA-immobilized CSCA-linkerE-DAE (8 U g-1 ) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L-1 d-allulose and 32.7 g L-1 D-fructose from 60 g L-1 inulin. CONCLUSION This study shed light on a feasible and cost-effective approach for the production of syrup containing d-allulose and D-fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. This article is protected by copyright. All rights reserved.